Project IV: Nef [unreadable] Hck interaction (T. Smithgall, Cores C, D, and E) - Large amounts of soluble Nef for preliminary NMR evaluation have been obtained. The regulatory region of Hck (Hck 32L, containing the SH3 and SH2 domains as well as the SH2-kinase linker), has been expressed in E. coli with very high yield. When combined in an equimolar ratio, a single, stable Nef-Hck32L complex is formed. Preliminary conditions for Nef-Hck crystallization have been identified using developed protocols. - Hck32L has been triple-labeled and ongoing NMR experiments are expected to reveal chemical shift changes within the SH3 domain and elsewhere upon Nef binding.